Use of minigene systems to dissect alternative splicing elements

Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.     

Locus control regions and epigenetic chromatin modifiers

Locus control regions are defined as gene regulatory sequences that enable chromosomal position-independent gene expression in transgenic mice. Recent studies have shown the ability of such regions to overcome the highly repressive effect of heterochromatin and have identified both trans-acting and cis-acting factors that participate in gene silencing and activation mechanisms.     

Allele-specific expression patterns reveal biases and embryo-specific parent-of-origin effects in hybrid maize

We employed allele-specific expression (ASE) analyses to document biased allelic expression in maize (Zea mays). A set of 316 quantitative ASE assays were used to profile the relative allelic expression in seedling tissue derived from five maize hybrids. The different hybrids included in this study exhibit a range of heterosis levels; however, we did not observe differences in the frequencies of allelic bias. Allelic biases in gene expression were consistently observed for approximately 50% of the genes assayed in hybrid seedlings. The relative proportion of genes that exhibit cis- or trans-acting regulatory variation was very similar among the different genotypes. The cis-acting regulatory variation was more prevalent and resulted in greater expression differences than trans-acting regulatory variation for these genes. The ASE assays were further used to compare the relative expression of the B73 and Mo17 alleles in three tissue types (seedling, immature ear, and embryo) derived from reciprocal hybrids. These comparisons provided evidence for tissue-specific cis-acting variation and for a slight maternal expression bias in approximately 20% of genes in embryo tissue. Collectively, these data provide evidence for prevalent cis-acting regulatory variation that contributes to biased allelic expression between genotypes and between tissues.     

In vivo activation of the intracrine vitamin D pathway in innate immune cells and mammary tissue during a bacterial infection

Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D(3) 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D(3) to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Subsequently, synthesis of 1,25(OH)(2)D(3) by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14(+) cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)(2)D(3), was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)(2)D(3) production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)(2)D(3) for local regulation of vitamin D responsive genes.     

Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing

All human genes contain a diverse array of cis-acting elements within introns and exons that are required for correct and efficient precursor messenger RNA (pre-mRNA) splicing. Recent computational analyses predict that most human exons contain elements required for splicing coinciding with an appreciation for the high frequency with which mutations that disruption pre-mRNA splicing cause disease. Minigenes provide a means to directly determine whether disease-causing mutations or single nucleotide polymorphisms (SNPs) affect splicing efficiency. Minigenes have also been instrumental in investigations of alternative splicing to identify cis elements required for cell-specific splicing events, demonstrating regulation of individual splicing events by specific RNA binding proteins, and correlating binding of these splicing regulators with splicing regulation. Here we present a versatile minigene plasmid vector designed for rapid cloning and analysis of cis elements and trans-acting factors that influence splicing efficiency or regulate cell-specific splicing. Ubiquitous expression and unique restriction sites allow for straightforward replacement of a variety of gene segments to analyze the effects of nucleotide substitutions on splicing, to identify tissue-specific regulatory elements, or to determine responsiveness to coexpressed proteins or small molecules.     

Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae.

The role of cis- and trans-acting elements in the expression of HIS4 has been examined by using HIS4-lacZ fusions in which lacZ expression is dependent upon the HIS4 5' noncoding region. The cis-acting sequences involved in regulation were defined by studying the effects of the wild-type and various deletions and their revertants on regulation via the general control of amino acid biosynthesis. The role of trans-acting genes was analyzed by studying the regulation of the HIS4-lacZ fusions in strains carrying mutations in the GCN (AAS) or GCD (TRA) genes and in strains carrying the GCN genes on high-copy-number plasmids. These studies have led to the following conclusions. (i) HIS4 is positively regulated by the general control. (ii) At least one copy of the 5'TGACTC3' repeat at -136 is required in cis for this regulation. (iii) Both the GCN4 gene and at least one copy of the repeated sequence are required for expression at the repressed level. (iv) The open reading frames in the 5' noncoding region are not required in either cis or trans for the regulation of HIS4.     

Evidence for redundancy but not trans factor-cis element coevolution in the regulation of Drosophila Yp genes.

In Drosophila melanogaster and the endemic Hawaiian species D. grimshawi three Yolk protein (Yp) genes are expressed in a similar sex- and tissue-specific pattern. In contrast, DNA sequence comparisons of promoter/enhancer regions show low levels of similarity. We tested the functional significance of these observations by transforming D. melanogaster with the genomic region that includes the divergently transcribed D. grimshawi DgYp1 and DgYp2 genes; we found that the introduced genes were expressed in female fat body and in ovaries but not in males. Moreover, we found D. grimshawi proteins in the hemolymph and accumulating in ovaries. Using reporter constructs we showed that the intergenic region from D. grimshawi was sufficient to drive accurate expression, but some low level of ectopic expression was seen in males. Transforming D. melanogaster with constructs bearing deletions within the D. grimshawi intergenic region revealed only subtle effects in the overall level of expression, suggesting a high level of redundancy. Testing mutants in the sex-specific regulator doublesex revealed that it is capable of repressing the DgYp genes in males. Together, these data show that D. melanogaster trans-acting factors can regulate the in vivo pattern of DgYp expression and support the notion of a redundant and complex system of cis-acting elements.     

Negative trans-acting factors extinguish Ia expression in B cell-L 929 somatic cell hybrids

Numerous studies have implicated trans-acting factors in the regulation of MHC class II gene expression. Some of these factors have been shown to act by inducing the expression of class II genes while others have been demonstrated to downregulate such expression. These reports have dealt almost exclusively with the role of trans-acting factors in the regulation of class II gene expression in hematopoietic-derived cells. We decided to extend these studies to the role trans-acting factors play in nonhematopoietic-derived (NHD) cells. In order to address this question we made somatic cell hybrids between the NHD Ltk- cell line and normal B cells to determine if the existence of positive trans-acting factors from the B cell would lead to the expression of Ltk- class II genes in the resultant hybrid. Our results clearly indicate that not only was there no induction of Ltk- class II gene expression in the hybrids, but there was a loss of B cell class II gene expression as well. These results suggest that Ltk- cells possess negative trans-acting factors that appear to predominate over the positive trans-acting factors possessed by B cells. We have further extended these studies to test the MHC-inducing activity of IFN-gamma and IL-4 on these hybrids. Our results indicate that the hybrids responded to IFN-gamma with an increase in class I but not class II expression for both fusion partners. Furthermore, neither B cell nor L cell class II genes were induced by IL-4. Taken together, these results indicate that Ltk- cells possess negative trans-acting factors that not only maintain the Ia- phenotype of these cells, but also block the action of positive trans-acting factors from B cells.     

Evolution of yellow gene regulation and pigmentation in Drosophila

BACKGROUND: Changes in developmental gene expression are central to phenotypic evolution, but the genetic mechanisms underlying these changes are not well understood. Interspecific differences in gene expression can arise from evolutionary changes in cis-regulatory DNA and/or in the expression of trans-acting regulatory proteins, but few case studies have distinguished between these mechanisms. Here, we compare the regulation of the yellow gene, which is required for melanization, among distantly related Drosophila species with different pigment patterns and determine the phenotypic effects of divergent Yellow expression. RESULTS: Yellow expression has diverged among D. melanogaster, D. subobscura, and D. virilis and, in all cases, correlates with the distribution of black melanin. Species-specific Yellow expression patterns were retained in D. melanogaster transformants carrying the D. subobscura and D. virilis yellow genes, indicating that sequence evolution within the yellow gene underlies the divergence of Yellow expression. Evolutionary changes in the activity of orthologous cis-regulatory elements are responsible for differences in abdominal Yellow expression; however, cis-regulatory element evolution is not the sole cause of divergent Yellow expression patterns. Transformation of the D. melanogaster yellow gene into D. virilis altered its expression pattern, indicating that trans-acting factors that regulate the D. melanogaster yellow gene have also diverged between these two species. Finally, we found that the phenotypic effects of evolutionary changes in Yellow expression depend on epistatic interactions with other genes. CONCLUSIONS: Evolutionary changes in Yellow expression correlate with divergent melanin patterns and are a result of evolution in both cis- and trans-regulation. These changes were likely necessary for the divergence of pigmentation, but evolutionary changes in other genes were also required.